Sandbox 201: Difference between revisions
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* <scene name='Sandbox_201/Atp_binding_site_grey/1'>ATP binding site</scene> | * <scene name='Sandbox_201/Atp_binding_site_grey/1'>ATP binding site</scene> | ||
:The N- and C-terminal domains are both able to form interactions with ATP and ATP analogues. But the ß-strands of the core region in the N-terminal domain contain most of the residues involved in binding ATP. <scene name='Sandbox_201/ | :The N- and C-terminal domains are both able to form interactions with ATP and ATP analogues. But the ß-strands of the core region in the N-terminal domain contain most of the residues involved in binding ATP. <scene name='Sandbox_201/Lys75_green_ball_v1/3'>Lys75</scene>, <scene name='Sandbox_201/Atp_binding_site_lys99/1'>Lys99</scene> (motif I), <scene name='Sandbox_201/Atp_binding_site_lys119/1'>Lys119</scene> (motif Ia), <scene name='Sandbox_201/Atp_binding_site_lys240/1'>Lys240</scene> (motif V), and <scene name='Sandbox_201/Atp_binding_site_lys242/1'>Lys242</scene> (motif V) interact with the phosphate groups of ATP and ATP analogues. | ||
:Lys99 is the site of adenylation in Rnl1, <ref>Thogersen, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. | :Lys99 is the site of adenylation in Rnl1, <ref>Thogersen, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. | ||
147, 325–329</ref> but in this structure this residue seems to be situated at a distance incompatible with covalent interaction with the phosphate of ATP (more than 3 Å).<ref name="main_article">K.El Omari, J.Ren, L.E.Bird, M.K.Bona, G.Klarmann, S.F.LeGrice, D.K.Stammers (2006) J. Biol. Chem. 281,1573-1579</ref> That could suggest that the formation of a covalent bond needs some conformational changes. But we do not know if a conformational change has to occur to allow the covalent bond formation, or if the formation of this bond leads to a conformational change. | 147, 325–329</ref> but in this structure this residue seems to be situated at a distance incompatible with covalent interaction with the phosphate of ATP (more than 3 Å).<ref name="main_article">K.El Omari, J.Ren, L.E.Bird, M.K.Bona, G.Klarmann, S.F.LeGrice, D.K.Stammers (2006) J. Biol. Chem. 281,1573-1579</ref> That could suggest that the formation of a covalent bond needs some conformational changes. But we do not know if a conformational change has to occur to allow the covalent bond formation, or if the formation of this bond leads to a conformational change. | ||
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:Metal ions are essential for the nucleotidyltransferase catalysis by all T4 DNA and T4 RNA ligases, which use the same two-metal ions mechanism. | :Metal ions are essential for the nucleotidyltransferase catalysis by all T4 DNA and T4 RNA ligases, which use the same two-metal ions mechanism. | ||
:The enzyme binds <scene name='Sandbox_201/Mg/1'>two magnesium ions</scene> Mg<sup>2+</sup>. The true substrate in the adenylation reaction is the ATP-Mg<sup>2+</sup> complex, <ref>Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704</ref> but nucleotidyltransferase enzymes cannot bind ATP-Mg<sub>2</sub> directly. They bind ATP-Mg first, then a second Mg<sup>2+</sup> ion. Each oh these cations <scene name='Sandbox_201/Mg_apc_residues/1'> | :The enzyme binds <scene name='Sandbox_201/Mg/1'>two magnesium ions</scene> Mg<sup>2+</sup>. The true substrate in the adenylation reaction is the ATP-Mg<sup>2+</sup> complex, <ref>Cherepanov, A. V., and de Vries, S. (2002) J. Biol. Chem. 277, 1695–1704</ref> but nucleotidyltransferase enzymes cannot bind ATP-Mg<sub>2</sub> directly. They bind ATP-Mg first, then a second Mg<sup>2+</sup> ion. Each oh these cations <scene name='Sandbox_201/Mg_apc_residues/1'>interact via hydrogen bonds</scene> with one phosphoryl oxygen from AMPcPP, three water molecules and two residues (Gly269 and Asp272), which both belong to the C-terminal domain. | ||
:The enzyme binds <scene name='Sandbox_201/Ca/1'>four calcium ions</scene> Ca<sup>2+</sup>. <scene name='Sandbox_201/Ca6_apc_residues/1'>Two</scene> are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246 via hydrogen bonds. They also interact with one phosphoryl oxygen of the AMPcPP. <scene name='Sandbox_201/Ca4_residues/2'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds. | :The enzyme binds <scene name='Sandbox_201/Ca/1'>four calcium ions</scene> Ca<sup>2+</sup>. <scene name='Sandbox_201/Ca6_apc_residues/1'>Two</scene> are coordinated to six water molecules. They do not directly interact with the enzyme, but via water molecules interacting with Glu227, Glu159, Lys99, Glu100, and Tyr246 via hydrogen bonds. They also interact with one phosphoryl oxygen of the AMPcPP. <scene name='Sandbox_201/Ca4_residues/2'>Two other</scene> are coordinated to four water molecules and interact with three enzyme residues (Ile211 and Asp212) via hydrogen bonds. | ||
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The T4 RNA ligase catalyzes the formation of phosphodiester bonds between the 5'-phosphate terminus of single-stranded nucleic acid (i) and the 3'-hydroxyl terminus of single-stranded nucleic acid (j). | The T4 RNA ligase catalyzes the formation of phosphodiester bonds between the 5'-phosphate terminus of single-stranded nucleic acid (i) and the 3'-hydroxyl terminus of single-stranded nucleic acid (j). | ||
ATP + ribonucleotide<sub>(i)</sub> + ribonucleotide<sub>(j)</sub> → AMP + diphosphate + ribonucleotide<sub>(i+j)</sub> | |||
